Engineering Biocatalysts for the C-H Activation of Fatty Acids using Ancestral Sequence Reconstruction

Selective, one-step C-H activation of fatty acids from biomass is an attractive concept in sustainable chemistry. Biocatalysis has shown promise for generating high-value hydroxy acids but to date enzyme discovery has relied on laborious screening and produced limited hits, which predominantly oxidise the sub-terminal positions of fatty acids. Here we show that ancestral sequence reconstruction (ASR) is an effective tool to explore the sequence-activity landscape of a family of multi-domain, self-sufficient P450 monooxygenases. We resurrected eleven catalytically active CYP116B ancestors, each with a unique regioselectivity fingerprint that varied from sub-terminal in the older ancestors to mid-chain in the lineage leading to the extant, P450-TT. In lineages leading to extant enzymes in thermophiles, thermostability increased from ancestral to extant forms, as expected if thermophily had arisen de novo. Our studies show that ASR can be applied to multi-domain enzymes to develop active, self- sufficient monooxygenases as regioselective biocatalysts for fatty acid hydroxylation.